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Revision as of 02:05, 17 October 2018
Methods
Contents
- Assembling the Pixcell Constructs
- Characterizing Pixcell Constructs
- Electrochemistry of Redox Modulators
- Spatial Electronic Control of Gene Induction
- Creating the Sox Library
- Characterising the Sox Library
- Testing Phenazine Methosulfate
- Biocontainment- Gp2 Growth Inhibition
- Fabric Bioprinting - Melanin
Experiments
Assembling the Pixcell Constructs
This protocol describes how to create the PixCell construct and PixCell construct with a degradation tag using Golden Gate assembly. Creating each of the constructs takes approximately two days .
pBR322 E. coli MG1655 genome DNA GFP Template DNA
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| Name | Template | Direction | Function | Tm (°C) | Sequence |
|---|---|---|---|---|---|
| PPC1 | MG1655 Genome |
Forward | Amplification of SoxRS regulon |
67.1 | CTAGTGGGTCTCCCTCGAGAGAAAGACAAAGACCGG |
| PPC2 | MG1655 Genome |
Reverse | Amplification of SoxRS regulon |
64 | GTCGATGGTCTCCCATAAATCTGCCTCTTTTCAGTG |
| PPC3 | GFP Storage Vector |
Forward | Amplification of GFP | 65 | GTCGATGGTCTCGTATGCGTAAAGGCGAAGAA |
| PPC4 | GFP Storage Vector | Reverse | Amplification of GFP | 68 | CTAGTGGGTCTCGGGACAGTAGCGAAAAAACCCCG |
| PPC5 | PixCell Construct | Forward | Addition of a degradation tag to GFP in the PixCell Construct |
65 | GCAAACGACGAAACTACGCTTTAGTAATGATACTAGAGCGCAAAAAACCCC |
| PPC6 | PixCell Construct | Reverse | Addition of a degradation tag to GFP in the PixCell Construct |
65 | CTAAAGCGTAGTTTCGTCGTTTGCCTTATACAGCTCGTCCATACCGTGG |
Characterizing Pixcell Constructs
This protocol describes how to measure E. coli growth and GFP expression over time with a plate reader, using the optical density at 600 nm as signal for cell density and excitation and emission wavelengths of 475. All characterization was performed using the FLUOstar BMG Labtech in greiner bioone F-bottom dark plates with clear bottom, to decrease fluorescence background. Each cycle of this experiment takes two days and has to be repeated two days, in order to have 2 biological and 2 technical replicates, for a total of 4 days and 4 96-well plates used.
- Autoclaved LB media
- Autoclaved 10x concentated LB media
- 500 uM pyocyanin stock solution (from Sigma-Aldrich, CAS: No. 86-66-5)
- 1250 mM potassium ferricyanide stock solution (from Sigma-Aldrich, CAS: No. 13746-66-2)
- 500 mM ferrocyanide stock solution (from Sigma-Aldrich, CAS 14459-95-1 )
- 2% Sodium Sulfite Solution
- Agar plates with colonies of E. coli DJ901 strains with respective constructs: PixCell Patterning Circuit 1 (BBa_K2862021). PixCell Patterning Circuit 2 (BBa_K2862022), negative control (DJ901 WT) and positive control (constitutively expressed GFP).
- 37°C shaking incubator
- BMG Labtech FLUOstar plate reader
- 4x Greiner BioOne 96-F 96-well microplate (black with clear bottom)
- 14 mL culture tubes
- autoclaved eppendorfs
Work in very sterile conditions. To prevent contaminations in the blanks, use triple antibiotics. Store pyocyanin and ferrocyanide and ferricyanide solutions in the freezer until use. We used 10x concentrated LB in order not to dilute nutrients at higher pyocyanin concentration; this was back diluted with autoclaved water. Mastemixes have double the working concentrations, as 100 uL of solution is then diluted with 100 uL of liquid cultures.
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- Grow starter liquid cultures of cells (Day 0, 10-15 min preparation + overnight)
- Prepare mastermixes (30 min)
- Fill 96-well plate with media solutions
- OD600 matching
- Add cells to 96-well plate
- Set up the script in the plate reader and start experiment
- Data analysis
Working in very sterile conditions, take 5 14mL culture tubes and prepare them according to table below:
| Label | E. coli strain | Construct | Antibiotic (Ab) | V Ab (uL) | V LB (mL) |
|---|---|---|---|---|---|
| PC1 | DJ901 | BBa_K2862021 | Kan + Amp | 5 + 5 | 5 |
| PC2 | DJ901 | BBa_K2862022 | Kan + Amp | 5 + 5 | 5 |
| - | DJ901 | none | Kan | 5 | 5 |
| + | DJ901 | GFP_Boo | Kan + Chl | 5 + 5 | 5 |
| Blanks | None | None | Kan + Amp + Chl | 5 + 5 | 5 |
With a pipette tip pick a desired colony* from an agar plate and release the contaminated tip in the culture tip. Place inoculated culture tubes in a 37° C in a shaking incubator overnight (for faster growth the angle between the vertical axis of the tube and the shaking plane of the incubator should be of 45°)
Mastermixes for each redox molecule concentration are prepared in 1.5 mL eppendorfs. The mastermixes are enough to fill 2 96-well microplates with 100 uL of solutions in each well, in order to have two replicate of the same experiment. Take 8 autoclaved 1.5 mL eppendorfs and prepare them as described in the table below.
Mastermizes for Day 1 and Day 2 :
| Condition | V Pyo (uL) | Kan (uL) | LB 10x uL | Autoclaved Water (uL) | Tot Vol (uL) |
|---|---|---|---|---|---|
| A = 0 uM Pyo | 0 | 2.44 | 122 | 1098 | 1220 |
| B = 0.01 uM Pyo | 0.0976 | 2.44 | 122 | 1097.9024 | 1220 |
| C = 0.025 uM Pyo | 0.244 | 2.44 | 122 | 1097.756 | 1220 |
| D = 0.05 uM Pyo | 0.488 | 2.44 | 122 | 1097.512 | 1220 |
| E = 0.1 uM Pyo | 0.976 | 2.44 | 122 | 1097.024 | 1220 |
| F = 0.25 uM Pyo | 2.44 | 2.44 | 122 | 1095.56 | 1220 |
| G = 0.5 uM Pyo | 4.88 | 2.44 | 122 | 1093.12 | 1220 |
| H = 1 uM Pyo | 9.76 | 2.44 | 122 | 1088.24 | 1220 |
Mastermixes for Day 3 and Day 4:
| Condition | V Pyo (uL) | Kan (uL) | LB 10x uL | Autoclaved Water (uL) | Tot Vol (uL) |
|---|---|---|---|---|---|
| A = 2.5 uM Pyo | 24.4 | 2.44 | 122 | 1073.6 | 1220 |
| B = 5 uM Pyo | 48.8 | 2.44 | 122 | 1049.2 | 1220 |
| C = 10 uM Pyo | 97.6 | 2.44 | 122 | 1000.4 | 1220 |
| D = 25 uM Pyo | 244 | 2.44 | 122 | 854 | 1220 |
| E = 50 uM Pyo | 488 | 2.44 | 122 | 610 | 1220 |
| F = 100 uM Pyo | 976 | 2.44 | 122 | 122 | 1220 |
Add 100 uL of media in the desired wells, according following the label on mastermixes and table below:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | PC1 | PC1 | BA | PC2 | PC2 | BA | - | - | BA | + | + | BA |
| B | PC1 | PC1 | BB | PC2 | PC2 | BB | - | - | BB | + | + | BB |
| C | PC1 | PC1 | BC | PC2 | PC2 | BC | - | - | BC | + | + | BC |
| D | PC1 | PC1 | BD | PC2 | PC2 | BD | - | - | BD | + | + | BD |
| E | PC1 | PC1 | BE | PC2 | PC2 | BE | - | - | BE | + | + | BE |
| F | PC1 | PC1 | BF | PC2 | PC2 | BF | - | - | BF | + | + | BF |
| G | PC1 | PC1 | BG | PC2 | PC2 | BG | - | - | BG | + | + | BG |
| H | PC1 | PC1 | BH | PC2 | PC2 | BH | - | - | BH | + | + | BH |
Once the cultures tubes in the shaking incubator appear cloudy, take them out and in a fresh microplate add 100 uL of each sample into a well. Add 100 uL of LB + antibiotic into a blank well. Take endpoint measurement of the filled wells and blank them with LB+antibiotic. Note down the OD600 value of the wells and dilute them to the desired initial OD600 (a value of 0.1 is suggested for a volume of 100 uL
Cell dilution calculator: Volume of LB+Antibiotic to add in culture tube = [( Vcells * ODcell)/desired OD] - Vcells E.g. to dilute 5mL of cells at an OD600 of 0.3 to OD of 0.1 = [ (5mL*0.3)/0.1 - 5] =10 mL
Pipette 100 uL of liquid cultures ot OD600= 0.1 into the corresponding wells,following 96-well microplate layout and culture tubes label.
For Pyocyanin experiments:
Set up a protocol that takes OD600 absorbance measurements and fluorescent intensity measurements every 10 minutes for 24 hours (288 cycles for 600 seconds cycles time).
For ferro/ferricyanide experiments:
For 200 uL of solution, we used a double orbital shaking rate of 200 rpm. Set temperature at 37°C.
Calculate mean and standard error for each set of replicates
Electrochemistry of Redox Modulators
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Spatial Electronic Control of Gene Induction
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Creating the Sox Library
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Characterising the Sox Library
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Testing Phenazine Methosulfate
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Biocontainment- Gp2 Growth Inhibition
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Fabric Bioprinting - Melanin
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